The current competing renewal encompasses our wet lab human studies directed at understanding the natural history and pathogenesis of Type 1 diabetes and applying that knowledge to develop improved prediction of the disease. In parallel, with other funding, we study the NOD mouse model, and the two sets of studies are intersecting with the hypothesis that insulin is a primary target of the autoimmunity that leads to beta cell destruction. As demonstrated by these studies, multiple genetic factors contribute to the appearance of islet autoantibodies in prospectively followed children including DR,DQ, and DP alleles, insulin gene, and PTPN22 R620W polymorphisms, while some loci have little influence (CTLA4). In the last funding period we discovered that in children followed from birth alleles DPB1*0402 and DRB1*0403 almost completely prevent islet autoimmunity in children less than 10 years old. In the last study period we also collaborated with Dr. Hutton who discovered the fourth major islet autoantigen (ZnT8) to define the predictive potential of ZnT8 autoantibodies. In children followed from birth we have recently discovered that mean levels of insulin autoantibodies inversely correlate with rate of progression to diabetes with no correlation for GAD65 or IA-2 autoantibodies. In addition, we have initial promising data for a non-radioactive plate capture insulin autoantibody assay that promises with its precision, sensitivity and specificity, to allow us to explore determinants of insulin autoantibodies in children developing diabetes, new onset patients, twins and even normal controls. We believe our MSD insulin autoantibody assay utilizing proinsulin can be improved but even with its current performance will likely replace fluid phase insulin autoantibody radioassays. An important specific aim is to fully characterize the MSD insulin autoantibody assay and to test modifying N-Hydroxy-succinamide labeling and biotinylation of proinsulin to further enhance the assay. Given new assay technology with high precision we are now in a position to explore determinants of multiple levels of insulin autoantibodies, (e.g. higher levels correlating with rate of progression to diabetes and low insulin inhibitable levels found in normal controls which may be potentially genetically determined. We will test the hypothesis that levels of insulin autoantibodies are correlated with abnormalities of T lymphocytes targeting insulin similar to our studies in the NOD mouse and specifically levels of insulin autoantibodies mark the rate of beta cell autoimmunity. These studies should improve our ability to design trials for the prevention of Type 1 diabetes as we better define the natural history of the disease.